Seminal fluid adipokinetic hormone increases insemination refractoriness in female Aedes aegypti

Aedes aegypti mosquitoes are the primary vector of several pathogens including dengue, Zika, and chikungunya viruses. Within an hour after an initial insemination, female Ae. aegypti are generally refractory to subsequent inseminations, a response that was first attributed in the 1960's to the effects of seminal fluid molecules (SFMs). Yet, despite the importance of this discovery, the actual molecules responsible for long-term female insemination refractoriness have remained unknown for 60 years. In a previous study, we identified adipokinetic hormone (AKH) precursor protein as an SFM in Ae. albopictus. AKH is a well-studied insect neuropeptide that impacts phenotypes including those related to metabolism, locomotion, and reproduction. In this study, we investigated whether AKH is an SFM in Ae. aegypti and whether it impacts female re-insemination patterns. We first established that AKH is produced and has enriched expression in the male reproductive tract. We then found that AKH is transferred to females during mating, and is, therefore, an SFM. Next, we generated an AKH-null line which allowed us to demonstrate that seminal fluid AKH contributes to long-term insemination refractoriness of females. Together, our findings (i) are the first to identify one of the seminal fluid proteins that influence long-term insemination refractoriness in Ae. aegypti and (ii) demonstrate a novel expression pattern and function for the well-studied, multi-functional adipokinetic hormone. These results lay the groundwork for understanding the evolution and mode of action of novel seminal fluid proteins as well as for investigating novel pathways or approaches for mosquito control.